Our goal is to investigate the impact of viral infecfion on alloreactivity on a human immune system. We will use humanized mice that develop a funcfional human immune system that can be infected with Epstein Barr Virus (EBV), lymphocytic choriomeningitis (LCMV), or influenza A (lAV), viruses associated with allograft rejecfion. In C57BL/6 mice, we have shown that heterologous immunity induced by LCMV infection leads to virus-specific allo-cross-reactive CDS T cells that can reject skin allografts and prevent tolerance induced by costimulation blockade. \Ne propose to test the hypothesis in humanized mice that virus infection leads to the generation of human virus specific allo-cross-reactive T cells that participate in rejection of human allografts. Humanized mice will allow analysis of these viruses using novel technologies to quantify naive and effector human alloreactive T cells and virus-induced allo-cross-reactive T cells. These analyses will permit direct determination of their ability to participate in allograft rejecfion. We also propose use of innovative siRNA in vivo delivery systems to block CD40-CD154 interaction. Aim 1 is to investigate the immune response to alloantigens in human immune system-engrafted NOD-sc/d lL2rf HLA-A2 mice transplanted with human islet or skin allografts. We will quantify the alloimmune response, determine kinefics of graft infiltration and allograft rejecfion, and establish models for studies of virus-induced heterologous immunity on allograft survival. Aim 2 is to quantify virus-specific and alloimmune responses during acute virus infection in humanized mice treated with cosfimulation blockade. We will determine human immune responses to EBV, LCMV, and lAV, and quantify virusspecific and allospecific T cells that develop as a consequence of heterologous immunity. We will test the hypothesis that virus infection generates heterologous immunity by inducing the development of virus-specific allo-cross-reactive T cells that participate in allograft rejection. These unique resources, novel technologies, and combined expertise in transplantafion, humanized mice, virology, and siRNA technology provide an opportunity to investigate the role of heterologous immunitv induced bv virus infection on alloreactivity and graft rejecfion in a human immune svstem.